Synthesis of novel β-propanamides to inhibit cholesteryl ester transfer protein (CETP)

Article abstract of DOI:10.1016/j.cclet.2016.10.016

A novel series of β-propanamide derivatives as inhibitors of cholesteryl ester transfer protein (CETP) were synthesized. Previously, H3 (IC₅₀2?μmol/L) was observed to inhibit CETP moderately (Xie et al., 2016). Structural modifications based on H3 led to discovery of the successful CETP inhibitor, known as 1-methyl-4-arylpyrazole. Using a similar approach, compound Q08 was identified as a highly potent CETP inhibitor with an IC₅₀of 490?nmol/L in vitro.

Full text of DOI:10.1016/j.cclet.2016.10.016

G Model
CCLET 3851 1–4
Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
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Original article
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Synthesis of novel
protein (CETP)
b
-propanamides to inhibit cholesteryl ester transfer
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Q1
Hong-Lei Xie, Chun-Chi Liu, Yi-Qun Li, Chang-Lin Bai, Chen-Zhou Hao, Jing Guo,
Chang-Qun Luo, Dong-Mei Zhao*, Mao-Sheng Cheng
6
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 6 June 2016
A novel series of
b
-propanamide derivatives as inhibitors of cholesteryl ester transfer protein (CETP)
mol/L) was observed to inhibit CETP moderately (Xie et al.,
2016). Structural modifications based on H3 led to discovery of the successful CETP inhibitor, known as
-methyl-4-arylpyrazole. Using a similar approach, compound Q08 was identified as a highly potent
Received in revised form 16 July 2016
Accepted 9 August 2016
Available online xxx
were synthesized. Previously, H3 (IC50 2 m
1
CETP inhibitor with an IC50 of 490 nmol/L in vitro.
ã 2016 Dong-Mei Zhao. Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of
Medical Sciences. Published by Elsevier B.V. All rights reserved.
Keywords:
CETP inhibitor
Cardiovascular disease
High-density lipoprotein
Low-density lipoprotein
In vitro
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1. Introduction
dalcetrapib (Scheme 1). Torcetrapib is the first inhibitor of phase III
clinical trials with a potent CETP IC50 of 50 nmol/L [13,14].
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Cardiovascular disease (CVD) is the leading cause of death in
industrialized and developing countries [1,2]. Epidemiological
studies have identified two key risk factors that increased the
likelihood of CVD events: decreased levels of high-density
lipoprotein-cholesterol (HDL-C) and increased levels of low-
density lipoprotein-cholesterol (LDL-C) [3–5]. Several clinical
studies have established the inverse relationship between
cardiovascular events and HDL-C serum levels [6–8]. Niacin
elevates HDL-C levels in clinical treatments but is not used often
due to considerable side effects [9,10]. Thus, there is a high demand
for a specific HDL-C raising therapies with better efficacy and
safety.
Plasma cholesteryl ester transfer protein (CETP) regulates the
inverse transport of cholesterol esters and also facilitates the
transfer of cholesteryl esters from HDL-C to both LDL-C and
very low density lipoprotein-cholesterol (VLDL-C) [11,12]. CETP
reduces atheroprotective HDL-C levels. Conversely, CETP inhibitors
help prevent retrograde cholesterol transport and therefore
increase HDL-C levels. Four small molecule inhibitors have entered
phase III clinical trials: torcetrapib, anacetrapib, evacetrapib and
However, torcetrapib was prematurely terminated due to a higher
mortality rate in the torcetrapib/atorvastatin group than the
atorvastatin group. Dalcetrapib also showed modest potency in
phase III clinical trials and was terminated because it failed to
exhibit a clinically relevant reduction in cardiovascular events [15].
Recently, potent CETP inhibitors anacetrapib and evacetrapib
entered phase III and overcame the issues with torcetrapib and
dalcetrapib [16,17]. Clinical trial data for both anacetrapib and
evacetrapib showed elevated HDL-C and lowered LDL-C without
side effects, such as torcetrapib.
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We previously reported a series of N,N-3-phenyl-3-benzyla-
minopropionanilide derivatives as CETP inhibitors. Structure–
activity optimization from screening L10 identified compound
H16 (IC50 0.15
mmol/L) as a potent CETP inhibitor and H3 (IC50
2
m
mol/L) as a modest CETP inhibitor [18]. The structure of H16
was notably flexible and might be the basis for its poor
bioavailability. To identify novel CETP inhibitors, we explored
b
-propanamide derivatives with low flexibility and reduced
synthetic complexity. To begin our optimization, we methodically
replaced -propanamide moieties of H3 with a focus on amide
b
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terminus substructure. Simultaneously, we focused on benzyl
moiety modifications with 3-1,1,2,2-tetrafluoroethoxyl and 4-F.
After further optimization of H3, we discovered compound
Q08 (IC50 490 nmol/L) to inhibit CETP in vitro, as measured by
BODIPY-CE fluorescence assay.
*
Corresponding author.
E-mail address: medchemzhao@163.com (D.-M. Zhao).
http://dx.doi.org/10.1016/j.cclet.2016.10.016
001-8417/ã 2016 Dong-Mei Zhao. Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights
reserved.
1
Please cite this article in press as: H.-L. Xie, et al., Synthesis of novel
Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.016
b-propanamides to inhibit cholesteryl ester transfer protein (CETP), Chin.

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CCLET 3851 1–4
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H.-L. Xie et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
Scheme 1. The four CETP inhibitors that have advanced to phase III clinical trials.
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2
. Experimental
Purity and homogeneity of the compounds were measured by
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chromatography on a glass column using silica gel (100–200 mesh)
assessed by TLC and HPLC chromatography. Mass spectra were
taken in ESI mode on an Agilent 1100 LC–MS (Agilent, Palo Alto, CA,
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2.1. Chemistry
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Compounds Q01–Q11 were prepared according to Scheme 2.
USA). NMR spectra were recorded at 400 MHz for H and 151 MHz
13
Overall, the compounds were prepared in three steps. The key
intermediate 2 was prepared by standard reductive amination
using commercially available benzaldehydes. The secondary amine
2
for 6 h to generate the key linker 3. Next, compound 3 was treated
with HOBT, EDCI and DIEA in DMF at 25 C for 2 h, followed by
substitution of various aliphatic amines or by condensation
reactions to obtain the desired target 5 compounds. Compound
or 101 MHz for C on a Bruker spectrometer with TMS as an
3 6
internal standard, CDCl or DMSO-d as solvent, and coupling
constants (J) were in hertz (Hz), and the signals were designated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet; br s, broad
singlet. These data can be found in the Supporting information.
ꢀ
was treated with acrylic acid through a Michael reaction at 50 C
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2.2. In vitro CETP inhibitory assay
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was prepared by aromatic amines with 1-methyl-4-(4,4,5,5,-
The CETP RP Activity Assay Kit (Catalogue # RB-RPAK; Roar)
uses a donor molecule containing a fluorescent self-quenched
neutral lipid that is transferred to an acceptor by CETP (Catalogue #
R8899; Roar). CETP-mediated transfer of the fluorescence neutral
lipid to the acceptor molecule results in an increase in fluorescence
(ExEm = 465/535 nm). Inhibition of CETP prevents lipid transfer
and therefore decreases the fluorescence intensity. The testing
compounds were entirely dissolved using 100% DMSO. Vibrating
hard on oscillator for more than 30 s helped significantly before
storing in a nitrogen cabinet. Stocking compounds (10 mmol/L)
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole through a Suzuki
reaction.
All solvents were purchased from Aladdin (Shanghai, China)
and were not purified further, and all of the chemicals were
purchased from commercial sources with purity >98%. Melting
points (mp) were determined in open capillaries on a Buchi
353 melting point apparatus (Buchi Labortechnik, Flawil,
Switzerland) and were uncorrected. Solvents used for moisture
sensitive reactions were distilled and used in an argon atmosphere.
ꢀ
Scheme 2. Synthetic routes. Reagents and conditions: (a) AcOH, substituted benzald ehydes; (b) NaBH
4
, r.t.; (c) 10% HCl, acrylic acid, 50 C; (d) substituted amines, HOBT,
EDCI, DIEA, r.t.
Please cite this article in press as: H.-L. Xie, et al., Synthesis of novel
Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.016
b-propanamides to inhibit cholesteryl ester transfer protein (CETP), Chin.

G Model
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H.-L. Xie et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
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Table 1
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were diluted with DMSO for eight titration points (1:5 serial
dilutions) in 96-well dilution plates. The assay was performed
according to the instruction for the CETP inhibitor screening kit
and recombinant CETP.
Structure–activity relationship (SAR) of
b-propanamides derivatives Q01–Q11.
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Compound
R
1
R
2
IC50
12.8
(mmol/L)
00
Q01
3-OCF
3-OCF
3-OCF
2
2
2
CF
CF
CF
2
H
2
H
2
H
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1
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. Results and discussion
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.1. In vitro activity and structure–activity relationships
Q02
Q03
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To evaluate the ability of various b-propanamides derivatives to
Q04
Q05
3-OCF
3-OCF
2
CF
CF
2
H
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inhibit CETP, all of these new synthetic compounds were assayed
using anacetrapib as a reference compound for their inhibitory
effects against CETP by BODIPY-CE fluorescence assay with CETP RP
Activity Assay Kit (Catalogue # RB-RPAK; Roar) (Table 1). Most of
the target compounds have a mild effect on CETP inhibition
activity, while several key structural changes indicated the general
SAR of this series. First, the effect of the substituted aniline
terminus on the inhibition of CETP was examined. Compound
2
2
H
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aniline termini substituted by 3-CF
and 3,4-diOCH exhibited modest CETP inhibition, while the one
substituted by 4-COOC compound Q06 exhibited an IC50 of
.8 mol/L. These results indicated that increased lipophilicity of
the aniline moiety did not lead to increased potency. Compounds
with 1-methyl-4-arylpyrazoleQ07 exhibited an IC50 of 0.72 mol/L.
When pyrimidines replaced benzene rings, we identified Q08,
which exhibits IC50 0.49 mol/L and lower than Q07. These
3 3
-4-Cl, 4-Br, 3-Cl, 3,5-bisCH
3
Q06
3-OCF
2
CF
2
H
2.8
2 5
H
2
m
m
m
Q07
Q08
4-F
4-F
0.72
0.49
suggested that increased hydrophilicity was important to improve
inhibition activity. The calculated LogP of Q07 and Q08 was 4.7
and 4.8, respectively. These results indicated that Q07 and Q08
physical properties fall within drug-like property space. However,
the activity of compound Q11 was higher than Q04, indicating that
compounds with 4-F benzyls exhibited higher potency than
compounds with 3-1,1,2,2-tetrafluoroethoxylbenzyl. These results
indicated that 4-F benzyl is significant to inhibit CETP.
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. Conclusion
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We report a novel CETP inhibitor based on the structure of our
previously developed compound H3. Most of the newly synthetic
compounds exhibited moderate inhibition CETP activity. Com-
pounds Q07 (IC50 0.72 mmol/L) and Q08 (IC50 0.49 mmol/L) were
identified to exhibit potent CETP inhibition. Compound Q08
titration is flexible and has a remarkable CLogP 4.8; therefore, Q08
was selected for further development. It is highly expected that
this novel scaffold will produce promising CETP inhibition agents
after further modifications.
Q09
Q10
Q11
4-F
4-F
4-F
7.2
18.5
5.3
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Acknowledgments
Q3 140
The work was supported by the National Natural Science
Foundation of China (No. 81373324) and the program for
Innovative Research Team of the Ministry of Education and
Program for Liaoning Innovative Research Team in University.
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Anacetrapiba
a
0.06
The positive control.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2016.10.016.
Please cite this article in press as: H.-L. Xie, et al., Synthesis of novel
Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.016
b
-propanamides to inhibit cholesteryl ester transfer protein (CETP), Chin.

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CCLET 3851 1–4
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H.-L. Xie et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
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Please cite this article in press as: H.-L. Xie, et al., Synthesis of novel
Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.016
b
-propanamides to inhibit cholesteryl ester transfer protein (CETP), Chin.